Materials

All materials used in this thesis are listed below. Waste was properly disposed according to safety guidelines.

2.1.1 Disposables

Disposable Reference Manufacturer

Bottle top filter, 0.22 µm SCGPT05RE Merck KGaA (Darmstadt, Germany) Cell culture dish, 10 cm (adherent) 83.3902 Sarstedt AG & Co. (Nümbrecht,

Germany)

Cell culture plate, 6-well (adherent) 83.3920.005 Greiner Bio-One GmbH (Frickenhausen, Germany)

Cell culture plate, 24-well (adherent) 83.3922.005 Greiner Bio-One GmbH Cell culture flask, T75 (adherent) 83.3911 Greiner Bio-One GmbH Cell culture plate, 6-well (suspension) 83.3920.500 Greiner Bio-One GmbH Cell culture flask, T75 (suspension) 83.3911.500 Greiner Bio-One GmbH

Cell scraper 83.1832 Sarstedt AG & Co.

Cell strainer, 40 µm 431750 Corning Inc. B.V. Life Sciences (Amsterdam, Netherlands) Cell strainer,100 µm 431752 Corning Inc. B.V. Life Sciences Corex tube, 38 mL 3117-0380 Thermo Fisher Scientific (Karlsruhe,

Germany)

Cover slip, 24x60 mm LH26.1 Carl Roth GmbH & Co. KG (Karlsruhe, Germany)

Cryo tube vial, 1.0 mL 37722 Thermo Fisher Scientific

DG8 gaskets 1863009 Bio-Rad Laboratories Inc. (Hercules,

USA)

DG8 cartridges 1864008 Bio-Rad Laboratories Inc.

Glass capillary 9201010 Hirschmann Laborgeräte (Eberstadt,

Germany)

Lint-free tissue 07730012 GVS Großverbraucherspezialisten eG

(Heidenheim, Germany) Pasteur pipette, 5 mL 86.1253.001 Sarstedt AG & Co.

Pasteur pipette, 10 mL 86.1254.001 Sarstedt AG & Co.

Pasteur pipette, 25 mL 760180 Greiner Bio-One GmbH

PCR plate, 96-well 951020389 Eppendorf AG (Hamburg, Germany) PCR Plate Heat Seal foil 1814040 Bio-Rad Laboratories Inc.

Reaction tube, 0.2 mL 0030124332 Eppendorf AG Reaction tube, 1.5 mL 0030125150 Eppendorf AG Reaction tube, 2.0 mL 0030120094 Eppendorf AG Serum sample tubes 41.1501.005 Sarstedt AG & Co.

Superfrost/plus slide, 75x25 mm 42409110 Glaswarenfabrik Karl Hecht GmbH & Co.

KG (Sondheim vor der Rhön, Germany) Syringe filter, PVDF membrane, 0.22 µm SLGV033RK Merck KGaA

Syringe filter, PVDF membrane, 0.45 µm SLHV033RB Merck KGaA

Tube, 30 mL 201150 Greiner Bio-One GmbH

Tube, 50 mL 227289 Greiner Bio-One GmbH

2 Materials and Methods

2.1.2 Instruments and accessories

Device Name Manufacturer

7T MRI system ClinScan Bruker BioSpin GmbH (Ettlingen,

Germany)

CCD Camera Color View III Soft Imaging Systems GmbH (Münster, Germany)

Centrifuge Megafuge 1.0R Sorvall Heraeus Instruments GmbH (Hanau, Germany)

Centrifuge Rotanta 460R Andreas Hettich GmbH & Co.KG

(Tuttlingen, Germany)

Centrifuge Sorvall RC-5C Plus

(HB-6 swinging bucket rotor)

Heraeus Instruments GmbH

Clinical chemistry analyzer COBAS Integra 400 plus

Roche Diagnostics Deutschland GmbH (Mannheim, Germany)

Cryo-microtome HM550 Thermo Fisher Scientific Microm

International GmbH (Walldorf, Germany) Digital camera FOcubusIEEE1394 PHASE GmbH (Lübeck, Germany) Droplet generator QX100 system Bio-Rad Laboratories Inc.

Droplet reader QX100 system Bio-Rad Laboratories Inc.

DNA migration chamber EASY Cast Electrophoresis system #B2

Owl Scientific Inc. (San Francisco, USA)

DNA migration chamber PerfectBlue Gelsystem Mini S

Peqlab Biotechnologie GmbH (Erlangen, Germany)

FACS analyzer BD FACS LSR

Fortessa

405, 488, and 561 nm Laser

BD Biosciences (Heidelberg, Germany)

FACS sorter BD FACS AriaIll

405, 488, and 561 nm Laser

BD Biosciences

Filter set DAPI U-MNU2 Olympus Europa SE & Co. KG

(Hamburg, Germany) Filter set Cerulean 438/24 BrightLine HC

483/32 BrightLine HC Beamsplitter HC BS 458

AHF analysentechnik AG (Tübingen, Germany)

Filter set GFP/Venus U-MNIBA2

Exciter: BP470-490 Dichroic: DM505 Emitter: BA510-550

Olympus Europa SE & Co. KG

Filter set mCherry U-MWIG2

Exciter: BP520-550 Dichroic: DM565 Emitter: BA580IF

Olympus Europa SE & Co. KG

Filter set Tripleband mCherry/Venus/Cerulean

Exciter:

CFP/YFP/mCherry ET Tripleband

Emitter:

ET Tripleband bs CFP/YFP/mCherry

AHF analysentechnik AG (Tübingen, Germany)

Fluorescence microscope IX81 Olympus Europa SE & Co. KG

Fluorometer Qubit 2.0 Thermo Fisher Scientific

Heating block Thermomixer 5436 Eppendorf AG

Heating plate PZ72 Harry Gestigkeit (Düsseldorf/Deutschland)

Incubator CB150 BINDER GmbH (Tuttlingen, Germany)

Laminar flow hood Herasafe Thermo Fisher Scientific

Light Microscope CKX41 Olympus Europa SE & Co. KG

Magnetic stirrer IKAMAG REO IKA-Werke GmbH & Co. KG (Staufen, Germany)

Spectrophotometer Nanodrop ND-1000 Thermo Fisher Scientific Spectrophotometer Qubit 2.0 Thermo Fisher Scientific Peristaltic pump XX8200230 Merck Millipore (Billerica, USA)

pH meter inoLab pH 720 WTW, Xylem Analytics Germany Sales

GmbH & Co. KG (Weilheim, Germany) Plate sealer QX100 system Bio-Rad Laboratories Inc.

Power supply Biometra Standard

Power Pack P25

Analytik Jena (Jena, Germany) Silicone hose, platin-hardened,

inner diameter 0.8 mm

OU-06371-00 Masterflex (Gelsenkirchen, Germany) Table top centrifuge 1. 5418

2. 5417R

Eppendorf AG

Thermocycler 1. Biometra

Professional 2. Biometra

Professional gradient

Analytik Jena

UV transilluminator 312 nm UV lamp PHASE GmbH

Vortex mixer REAX control Heidolph Instruments GmbH & Co. KG (Schwabach, Germany)

Water bath Typ 1008 GFL Gesellschaft für Labordiagnostik

GmbH (Burgwedel, Germany)

Water purifier membraPure membraPure GmbH (Henningsdorf,

Germany)

2.1.3 Software

Software Version Manufacturer

BioEdit Sequence Alignment Editor v7.2.5 Tom Hall Ibis Therapeutics (Carlsbad, USA)

FACSDiva v4.2.2 BD Biosciences

FireCam Control software v2.03 PHASE GmbH

Fire Wire Soft Imaging Systems GmbH

GraphPad Prism v6.0 STATCON (Witzenhausen, Germany)

ImageJ v1.51j8 Wayne Rasband, National Institutes of

Mental Health (Bethesda, USA)

K-PACS v1.6.0 Image Information Systems Ltd. (London,

United Kingdom)

Microsoft Excel 2007 Microsoft Corporation (Redmond, USA)

Microsoft Word 2007 Microsoft Corporation

Microsoft Powerpoint 2007 Microsoft Corporation Microsoft Image Composite Editor v2.0.3 Microsoft Corporation

2 Materials and Methods

Nanodrop ND-1000 v3.7.1 Thermo Fisher Scientific

pDRAW32 1.0 Revision v1.1.133 AcaClone Software (Copenhagen, Denmark)

Primer Express v3.0.1 Applied Biosystems by Thermo Fisher

Scientific

QuantaSoft, Inc. v1.7.4.0917 2014 Bio-Rad Laboratories Inc.

Qubit v3.10 Thermo Fisher Scientific

SnapGene Viewer v3.3.3 GSL Biotech LLC (Chicago, USA)

2.1.4 Chemicals and reagents

Chemical Reference Manufacturer

Acetone 9002-02 J.T. Baker Avantor Performance Materials

B.V. (Deventer, Netherlands)

alpha-D-glucose 158968 Sigma-Aldrich (München, Germany)

Agarose Ultra Pure Agarose 16500500 Thermo Fisher Scientific

Ampicillin HP62.1 Carl Roth GmbH & Co. KG

β-mercaptoethanol M3148 Sigma-Aldrich

CaCl2 1.02820.1000 Merck KGaA

ddPCR Droplet Reader Oil 1863004 Bio-Rad Laboratories Inc.

ddPCR Supermix for Probes 1863010 Bio-Rad Laboratories Inc.

DNA loading dye (6x) R0611 Thermo Fisher Scientific

DNA/RNA-dye, peqGREEN 732-3196 Peqlab/VWR International GmbH (Hannover, Germany)

dNTPs 10 µM each: dATP; dCTP;

dGTP; dTTP

R0142; R0151 R0161; R0171

Thermo Fisher Scientific Droplet generation oil for probes 1863005 Bio-Rad Laboratories Inc.

EGTA E4378 Sigma-Aldrich

GeneRuler 1 kb plus DNA Ladder (0.1 µg/µL)

SM1343 Thermo Fisher Scientific

KCl 6781 Carl Roth GmbH & Co. KG

LBAmp agar (Fast Media Amp agar) fas-am-s InvivoGen (San Diego, USA)

LB medium 6669 Carl Roth GmbH & Co. KG

MgCl2 AM9530G Thermo Fisher Scientific

Methylbutane 721123 Sigma-Aldrich

NaCl (solid) 3624-01 J.T. Baker

Na2HPO4 T876 Carl Roth GmbH & Co. KG

Na2HPO4 • H2O T877 Carl Roth GmbH & Co. KG

NaOH pellets 106482 Merck KGaA

Paraformaldehyde* 158127 Sigma-Aldrich

Sucrose** US1SX1075/1 Merck KGaA

UltraPure DNA Typing Grade 50X TAE (Tris/Acetate/EDTA) Buffer, diluted in deionized H2O to 1x TAE

24710030 Thermo Fisher Scientific

Tissue-Tek Cryomold Intermediate embedding forms

4566 Sakura (Torrance/USA)

Tissue-Tek O.C.T. Compound 4583 Sakura (Alphen aan den Rijn, Netherlands)

*For complete dissolution of the paraformaldehyde powder, the flask was incubated for 1.5 h at 60°C and shaken occasionally. As soon as the solution was cooled down to RT it was ready to use. For storage, the solution was aliquoted in 50-mL tubes and kept at -20°C.

**The 20% sucrose solution was prepared with double distilled water and sterile filtered through a 0.22-µm filter unit (Merck Millipore) under the hood. For storage, the solution was kept at 4°C.

2.1.5 Kits

All listed kits were used according to manufacturers‟ instructions.

Kit name Reference Manufacturer

DAKO EnVision⁺ Dual Link System-HRP (DAB⁺) Kit

K406511-2 Agilent Technologies Deutschland GmbH (Waldbronn, Germany) PureLink HiPure Plasmid Maxiprep Kit K210007 Thermo Fisher Scientific

QIAamp DNA Blood Mini Kit 51106 QIAGEN GmbH (Hilden, Germany) QIAGEN AllPrep DNA/RNA Mini Kit 80204 QIAGEN GmbH

QIAprep Spin Miniprep Kit 27106 QIAGEN GmbH

QIAquick Gel Extraction Kit 28706 QIAGEN GmbH

QIAquick PCR Purification Kit 28106 QIAGEN GmbH

Qubit dsDNA BR Assay Kit Q32850 Thermo Fisher Scientific Qubit dsRNA HS Assay Kit Q32852 Thermo Fisher Scientific

2.1.6 Enzymes and oligos

Enzyme Reference Manufacturer

EcoRI Fast Digest FD0274 Thermo Fisher Scientific

FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)

EF0651 Thermo Fisher Scientific

Liberase TM Research Grade 05401119001 Roche Diagnostics Deutschland GmbH

NotI Fast Digest FD0593 Thermo Fisher Scientific

Phusion High-Fidelity DNA Polymerase (2,000 U/mL)

M0530 New England Biolabs (Frankfurt am Main, Germany)

T4 DNA Ligase (5 U/µL) EL0011 Thermo Fisher Scientific

All oligonucleotides such as primers and probes were purchased from Eurofins mwg. Primers were ordered from Eurofins Genomics (Ebersberg, Germany) choosing “Salt Free” or HPLC purification method. Used oligonucleotides are listed in Supplementary Tables S1 and S2.

2.1.7 Media and additives for cell culture

Product Reference Manufacturer

Chloroquine PHR1258 Sigma-Aldrich

DMEM, high glucose, GlutaMAX Supplement, pyruvate

31966021 Thermo Fisher Scientific DPBS (1x) no calcium, no magnesium 14190094 Thermo Fisher Scientific

Fetal Bovine Serum F7524 Sigma-Aldrich

L-Glutamine 25030149 Thermo Fisher Scientific

Hepatocyte Wash Medium 17704024 Thermo Fisher Scientific

HepatoZYME-SFM 17705021 Thermo Fisher Scientific

HEPES (1M) 15630056 Thermo Fisher Scientific

Penicillin-Streptomycin (100 IU/mL/100 µg/mL)

15140122 Thermo Fisher Scientific

Polybrene TR-1003 Sigma-Aldrich

Trypan Blue Solution (0.4%) 15250061 Thermo Fisher Scientific Trypsin-EDTA (0.05%), phenol red 25300054 Thermo Fisher Scientific

2 Materials and Methods

2.1.7 Buffer for viral vector production 2x HBS (HEPES buffered saline)

275.8 mM CaCl2

10.2 mM KCl 1.41 µM NaH2PO4

42 mM HEPES

1.1 mM alpha-D-glucose

ad 1L with ddH₂O; pH 7.05 adjusted by 1 N NaOH and sterilized via 0.22 µm filtration, storage at -20°C

2.1.8 Buffer and stock solutions for liver perfusion and hepatocyte isolation

All buffers were adjusted to pH 7.4 with 1 N NaOH, sterile filtered with the BottleTop-filter system (0.22 µm), and stored at 4°C. Prior to use, the buffers were prewarmed to 39°C.

10x Leffert‟s stock solution 100 mM HEPES

30 mM KCl 1.3 M NaCl

10 mM NaH₂PO₄•H₂O 100 mM alpha-D-Glucose ad 1000 mL ddH₂O

EGTA stock solution

100 mL 10x Leffert„s stock solution 5 mM EGTA

ad 1000 mL ddH2O

Calcium chloride stock solution 63 mM CaCl₂

ad 500 mL ddH2O

Washing buffer

45 mL 10x Leffert‟s stock solution

50 mL EGTA stock solution (final 0.5 mM) ad 500 mL ddH2O

Collagenase buffer

50 mL 10x Leffert‟s stock solution

5 mL CaCl2 stock solution (final 0.63 mM) ad 500 mL ddH₂O

2.1.9 Surgical kit

All material used in hepatocyte transplantation surgery is listed below.

Product Reference Manufacturer

26 G cannula; BD Microlance 3 1/2;

0.45x13 mm

305111 Becton Dickinson (Heidelberg, Germany) 27 G cannula; BD Microlance 3 3/4;

0.4x13 mm

302200 Becton Dickinson

Carprofen 50 mg/mL Rimadyl 400684.00.00 Pfizer Deutschland GmbH (Berlin, Germany)

Enrofloxacin (Baytril 2.5% oral solution)

3572731 Bayer Vital GmbH (Leverkusen, Germany)

Forene inhalation solution (active agent: isofluran)

10182054 Abbvie Deutschland GmbH & Co. KG (Wiesbaden, Germany)

Glass syringe 100 µL Model 1710 RN SYR, NDL, barrel inner diameter 1.46 mm, barrel outer diameter 7.75 mm; Needle G 28 /15mm/

pointstyle

7803-02 Hamilton by Sigma-Aldrich

Infusion set Vasofix Safety PUR, 22G, 0.9x25 mm

4269098S-01 B. Braun Melsungen AG (Melsungen, Germany)

Insulin syringe, 0.33 mm, 29G, U100 305932 Becton Dickinson

Ketamin 100 mg/mL 9089.01.00 Wirtsgenossenschaft deutscher Tierärzte eG (Garbsen, Germany)

Novaminsulfon (500 mg/mL) 08713863 Ratiopharm GmbH (Ulm, Germany) Octenisept, alcohol-free 173702 Schülke & Mayr (Norderstedt, Germany) Ophthalmic ointment (Bepanthen,

Augen- und Nasensalbe)

01578681 Bayer Vital GmbH PGA Resorba needle DS 18, 3/0

USP, 0.45 m

PA 11416 RESORBA Medical GmbH (Nürnberg, Germany)

PGA Resorba suture 3/0, 0.45 m0 PS 465 RESORBA Medical GmbH

Povidone-iodine unguent 04923204 Mundipharma GmbH (Limburg, Germany) Sedaxylan 20 mg/mL 400650.00.00 Wirtsgenossenschaft deutscher Tierärzte

eG (Garbsen, Germany) Wound Clip (MikRon Autoclip, 9 mm) 427631 Becton Dickinson

Wound Clip Applier 427630 Becton Dickinson

Wound Clip Remover 427637 Becton Dickinson

2 Materials and Methods

Im Dokument Hepatocellular carcinoma : Establishing a new transplantation model for the study of benign and malignant clonal regeneration of the murine liver in vivo (Seite 34-41)